Project Summary/Abstract Subunit vaccines are a key strategy for preventing infectious disease and related deaths.. However, the use of subunit antigens, which elicit a weaker immune response than intact pathogen, has coincided with a need for safe and effective adjuvants to improve vaccine efficacy, increase immune response, and reduce the size and quantity of vaccine necessary to impart immunity. However, we lack a detailed picture of how engaging multiple innate immune receptors simultaneously or sequentially, on the same cell, or different cells, alters the outcome of the immune response. Our research team has engineered a new, flexible, pathogen-mimicking recombinant outer membrane vesicle (rOMV) platform capable of presenting functional antigen together with desired combinations of adjuvants. We intend to use this innovative technology to investigate how different adjuvant combinations of TLR and NOD agonists engage the innate immune system and help direct the immune response against viral and bacterial pathogens. Our hypothesis, to be evaluated through the Specific Aims, is that rOMVs can co-present antigen with specific combinations of adjuvants to induce pathogen-matched and protective immune responses (e.g., anti-viral and anti-bacterial). The specific aims are to: Aim 1: Identify the molecular pathways elicited by individual or combinations of adjuvants on rOMVs. rOMVs will be decorated with TLR and NOD agonists to understand how the resulting innate immune receptor agonists activate and influence cell signaling pathways using established reporter cells, and determine how those pathways are altered by co-presentation of TLR and NOD agonists. We will compare the response to soluble combinations of the same adjuvants on both signaling and dendritic cell activation and maturation. Aim 2: Determine how rOMV-adjuvant combinations direct the adaptive immune response to immunization using OVA as a model antigen. We will use OVA antigen to characterize the adaptive immune response induced by rOMV expressing single or combinations of adjuvants and compare them to single or combinations of soluble adjuvants. We will identify the combination(s) that promote strong and directive T cell, B cell and/or memory responses. Aim 3: Determine whether tailored rOMV adjuvant combinations can direct specific anti-viral and anti-bacterial protective immunity. We will use OVA-presenting rOMVs, as well as pathogen-specific antigens presented on rOMVs, decorated with different single or combined adjuvants in challenge models for both a viral (influenza) and a bacterial pathogen (Listeria) modified to express OVA as well as the native pathogens (i.e., no OVA). We will examine how innate signaling pathway activation and immune response correlate to protective immunity. The anticipated outcome of the proposed studies is a mechanistic understanding of how adjuvant combinations work at the molecular, cellular and organismal level to protect against viral and bacterial pathogens. The outcome will help to define a set of guiding principles that we and other investigators in the field can use to more robustly design adjuvant systems to maximize the efficacy of subunit vaccines.